Antisense nucleic acids targeting HBV

ABSTRACT

The present invention concerns antisense nucleic acids which are directed against specific sequences of RNA molecules originating from HBV and vectors and host cells containing these antisense nucleic acids.

The present application claims the benefit of priority from PCTApplication Serial No. PCT/EP98/03632 (WO 98/58055) filed Jun. 16, 1998,and German Application Serial No. 197 25 803.4 filed Jun. 18, 1997.

The present invention concerns antisense nucleic acids, which aredirected against specific sequences of RNA molecules originated fromHBV, and vectors and host cells containing these antisense nucleicacids.

Chronic infections with the hepatitis-B virus (HBV) play a central rolein the pathogenesis of liver cell carcinoma in humans. Hepadna viruses,of which HBV is one, replicate their DNA genome through reversetranscription of an RNA intermediate, the so-called pregenomic RNA. Theapplication of antisense inhibitors represents, in a particularlyspecific manner, a therapeutic possibility for HBV infection. Theinhibition of the propagation of the HBV can take place with the help ofthe antisense strategy both through inhibition of reverse transcription,with pregenomic RNA as the target, as well as through blocking oftranslation, with viral mRNAs as targets. Pregenomic RNA represents themost reasonable RNA target since it is the earliest target for anantisense nucleic acid in the replication cycle of the virus.

In the past, HBV-directed antisense sequences were directed arbitrarilyin particular against 5′ regions of viral mRNAs, their cap structure, orthe polyadenylation signal (Goodarzi et al., J. Gen. Viro. 71: 3021-3025(1990); Blum et al., Lancet 337: 1230 (1991); Wu, G. Y. and Wu C. H., J.Biol. Chem. 267: 12436-12439 (1992); Reinis et al., Folia Biol. (Praha)39: 262-269 (1993); Yao et al., Zhanghua Yi Xue Za Zhi 74: 74-76 (1994);Yao et al., Acta Virol. 39: 227-230 (1995); Yao et al., J. Virol. Hepat.2: 85-89 (1995); Moriya et al., Biochem. Biophys. Res. Comm. 278:217-223 (1996)).

Since the selection of the antisense sequences in the past has beenrather arbitrary and without rational selection criteria, a large partof the sequences investigated were not effective. Important influencevariables on the association behavior between the antisense nucleic acidand the target RNA, such as, for example, the accessibility of thetarget RNAs to the antisense nucleic acids, were not considered.Furthermore, the structural diversity of the antisense RNAs was notconsidered and thus the potential of effective antisense inhibitors wasnot exhausted.

Thus the present invention is based on the object of preparing antisensenucleic acids which hybridize particularly effectively with thepregenomic RNA of human HBV in particular.

This object is realized through the embodiments characterized in theattached patent claims. In particular, an antisense nucleic acid isprepared which is directed against a specific sequence of an RNAmolecule originating from HBV as target molecule with the antisensenucleic acid being smaller than the RNA molecule and having anassociation constant k of at least 5×10³ M⁻¹s⁻¹. Preferably theassociation constant k is at least 2×10⁴ M⁻¹s⁻¹.

The concept “antisense nucleic acid” means a native, semi-synthetic,synthetic, or modified nucleic acid molecule of deoxyribonucleotidesand/or ribonucleotides and/or modified nucleotides.

The RNA molecule originating from HBV can be pregenomic RNA, pre-C mRNA,pre-S1 mRNA, pre-S2/S mRNA, or the transcript of the X gene. Preferablythe RNA molecule is the pregenomic RNA of HBV. Pre-C mRNA is an RNAtranscript initiated at the precore initiation codon of the C geneencoding the nucleopsid protein of HBV. Pre-S1 mRNA is an RNA transcriptinitiated at the pre-S1 initiation codon of the S gene encoding the“major” envelope protein HbsAg of HBV. Pre-S1 mRNA is an RNA transcriptinitiated at the second, or S1, initiation codon of the S gene encodingthe “major” envelope protein of HbsAg of HBV. See Fauci et al., ed.Harrison's Principles of Internal Medicine, McGraw-Hill, 14^(th) Ed. Pp.1677-1678.

In a preferred embodiment of the present invention, the antisensenucleic acid contains the antisense oligodeoxynucleotide designated withSEQ ID No. 1 to 3. In another preferred embodiment of the presentinvention, the antisense nucleic acid contains the antisense ribonucleicacid sequences designated with SEQ ID No. 4 through 121 which rapidlyhybridize with the pregenomic RNA.

The preferred antisense nucleic acid sequences, designated with SEQ IDNo. 1 through 121, hybridize in vitro with above-average rapidity withthe pregenomic RNA and are therefore excellent inhibitors of HBVreplication in vivo. Surprisingly it was found that these preferredantisense nucleic acids have a selectivity with respect to pregenomicRNA of HBV even if identical target sequence regions occur on the mRNAsof HBV.

A further object of the present invention is a vector which contains theabove defined antisense nucleic acid according to the invention or whichcontains a corresponding DNA sequence complementary to the antisensenucleic acid which following transcription in suitable host cellsresults in the above-defined antisense nucleic acid according to theinvention. The vector according to the invention can preferably containsuitable regulatory elements such as promoters, enhancers, andtermination sequences. In an embodiment according to the invention, thevector can be used, for example, for stable integration of the nucleicacid according to the invention into the genetic material of a hostcell.

A further object of the present invention is a host cell which containsthe antisense nucleic acid or the vector according to the invention.Suitable host cells are, for example, all mammalian cells which carrysequences of hepatitis viruses, preferably human cells which carryhepatitis B virus sequences.

A further object of the present invention is a medication which containsthe antisense nucleic acid or the vector according to the invention,possibly in a pharmaceutically acceptable base and/or diluting agent.The medication according to the invention can be used to inhibit oreliminate disease conditions caused by HBV infections through transientor stable integration of the antisense nucleic acid according to theinvention by transformation and/or transfection in host cells infectedwith HBV.

DESCRIPTION OF THE FIGURES

The drawings show the following:

FIG. 1 is a schematic representation of the in vitro selection tests andthe identification of rapidly hybridizing antisense RNA molecules.

FIG. 2 is a photographic representation of an autoradiograph which showsthe identification of Sau5-1 derived, rapidly hybridizing antisense RNAmolecules.

FIG. 3 is a photographic representation of an autoradiograph which showsthe identification of Sau3 derived, rapidly hybridizing antisense RNAmolecules.

FIG. 4 is a graphic representation which shows the chain-lengthdependence of the association constants of Sau5-1 derived antisense RNAmolecules.

FIG. 5 is a graphic representation which shows the chain-lengthdependence of the association constants of Sau3 derived antisense RNAmolecules.

The present invention will be explained in greater detail using thefollowing example.

EXAMPLE Plasmids for the In Vitro Synthesis of Antisense RNAs and TargetRNA

Conventionally obtainable, cloned HBVadw2-DNA was cleaved with therestriction enzyme Sau3Al and the restriction fragments were cloned inthe BamH1 cleavage site of the transcription vector pGEM-7Zf+(Promega)and confirmed through the DNA sequencing. Two of these clones, pSau3 andpSau5-1, contained strands of HBVadw2 (pSau3: 334 nucleotide, positions944-1266; pSau5-1: 260 nucleotides, positions 29-288) in antisenseorientation with respect to the SP6 promoter. The derived antisense RNAs(Sau3 and Sau5-1) of these constructs contain additional polylinkersequences at their 5′ ends (41 nucleotides) and their 3′ ends (6nucleotides). The RNA strand which contains the complementary sequencesof Sau3′ and Sau5-1 was transcribed in vitro using the SP5-RNApolymerase. The sense transcript contains 3320 of 3321 nucleotides ofthe pregenomic RNA (positions 2-3321) and additional unspecificsequences at the 5′ end (3 nucleotides) and at the 3′ end (209nucleotides).

In Vitro Transcription of RNA

The plasmids were linearized prior to the in vitro transcription by thefollowing enzymes: pSau3 and pSau5-1 by HindIII, PSP6HBV by PvuII.SP5-RNA-polymerase (Boehringer Mannheim) was used for in vitrotranscription. 5 μg each of linearized template RNA was incubated in areaction buffer which contained 40 mM tris-HCl, pH 8.0 (20° C.), 6 mMMgCl₂, 10 mM DTT, and 2 mM spermidin, in a total volume of 200 μl at 37°C. for 90 min. The reaction was started through addition of 20 U T7 RNApolymerase and was halted through addition of 10 mM MgSO₄ and 20 UDNasel (Rnass-free, Boehringer Mannheim). After an additional incubationfor 15 min at 37° C., the DNasel decomposition was halted throughaddition of 25 mM EDTA. The RNAs were purified through gel filtration(Sephadex G50 medium, Pharmacia), phenol extraction, and ethanolprecipitation.

³²P Labeling of RNAs

The 5′ ends of the in vitro transcribed antisense RNA molecules (10 ng)were ³²P-marked with phosphatase from calf intestine and with 100 μCi[γ-32F]-ATP (10.0 mCi/mi, 3000 Ci/mmol) and polynucleotide kinase(Boehringer Mannheim) according to the methods known in the state of theart.

Alkaline Hydrolysis of Antisense RNA Molecules

For generation of a variable pool of HBV-directed antisense RNAmolecules, ³²P-5′-labeled original RNA was successively shortenedthrough limited alkaline hydrolysis according to methods known in thestate of the art. In this process, 2.5 pmol 5′-labeled RNA molecules inTE buffer (10 mM Tris/HCl, pH 8.0, 1 mM EDTA) was heated with 1.5volumes of 0.5 mM NaHCO₃ to 96° C. for 8-10 min and then cooled on iceand desalted through gel filtration (sephadex G-50 fine, Pharmacia).After ethanol precipitation, the RNA molecules were dissolved in TEbuffer. Following this the variable mixture of RNA molecules was heatedfor 10 min to 75° C. and was slowly cooled to 37° C. before use inhybridization tests in order to obtain a native folding of the RNAmolecules.

Kinetic In Vitro Selection Method for Selective Identification ofRapidly-Hybridizing Antisense RNA Molecules

The experimental process is described in the state of the art and isschematically represented in FIG. 1. 5′-labeled and partially hydrolyzedRNA was incubated with a ten or twenty fold molar excess (25 or 50 pmol,conditions of pseudofirst reaction order) of HBV pregenomic RNA in anend volume of 60 μl and with approximately physiological salt conditions(100 mM NaCl, 20 mM Tris/HCl, pH 7.4 and 10 mM MgCl₂) at 37° C. in orderto favor the selection of physiologically active molecules. During thehybridization, aliquots of the reaction mixture were removed at variouspoints in time and were introduced into ten volumes of precooled stopbuffer (7 M urea, 20 mM Tris/HCl, pH 8.0, 10 mM EDTA, 0.5% SDS, 0.04%bromophenol blue, 0.04% xylene cyanol). After separation by means ofnative agarose-gel electrophoresis (0.8% agarose, TBE) the single-strandRNA molecules and the double-strand RNAs formed were cut from the geland were recovered through centrifuging the gel pieces according to the“freeze-thaw method” (liquid nitrogen/37° C.). After the phenoltreatment and ethanol precipitation, the RNA was dissolved in stopbuffer and the single-strand as well as hybridized fractions wereanalyzed by means of denaturing PAGE (10% PAA, 7 M urea, TBE). The gelswere dried and exposed to an x-ray film or a phosphorimager screen forfurther computer-aided quantitative analysis.

Determination of the Association Constants of Individual Antisense RNAMolecules

For the quantitative analysis of the band intensities, the dried PAAgels were tested using a phosphorimager (Molecular Dynamics). Using theprogram “Image Quant” (Molecular Dynamics), the band intensities weremeasured and the data was transferred to the program “EXCEL”(Microsoft). For individual RNA molecules, the band intensities wereentered on the time axis and a curve for an individual exponentialdecomposition was fitted through non-linear regression using the program“GRAFIT” (Erithacus Software, London, Great Britain). By plotting thecalculated association constants (calculation based on the conditions ofthe pseudofirst reaction order) against the length of the antisense RNAmolecules, different groups of rapidly hybridizing antisense RNAmolecules were detected.

Antisense RNA, Directed Against the Pregenomic RNA of Human HBV Typeadw2

Two original antisense RNA molecules which are directed againstdifferent regions of the pregenomic RNA of HBV were selected forperforming an in vitro selection test. The first target area, positions29-288, which is sequence homologous with regions of the 5′ ends of thepreS1 and preS2/S mRNAs, was already known as an effective target ofantisense nucleic acids and was selected as a result of its high foldingenergy of the RNA secondary structure. The second target area, positions933-1266, was selected on the basis of its sequence homology with the 5′end of the X gene transcript. The pregenomic RNA is approximately 10times longer than these two antisense RNA molecules. As a result, thesingle-strand fraction can be separated in the course of the in vitroselection test from the double-strand fraction.

Kinetic Selection and Identification of Rapidly Binding ComplementaryRNA

The original antisense RNAs were synthesized through in vitrotranscription and ³²P labeling at the 5′ end. Limited alkalinehydrolysis resulted in a pool of antisense RNA molecules which had the5′ end in common, but had a successively shortened 3′ end. Theassociation constant for these molecules was measured at 37° C. andphysiologic ion concentration. The in vitro selection and identificationof rapidly hybridizing antisense RNA molecules is schematically depictedin FIG. 1. The rapidly hybridizing molecules are the first whichdisappear from the trace of single-strand fraction and can be detectedthrough their chain length. The experimental identification of rapidlybinding, complementary RNA molecules is shown in FIGS. 2 and 3. Theassociation constants, in relation to the chain length of relatedmolecules which stem from one of the two original antisense constructs,are shown in FIGS. 4 and 5. The maximum association constants reachvalues of 2 to 4×10⁴ M⁻¹s⁻¹, and even for relatively small antisense RNAmolecules, they are higher than for the original full-length RNA and 3times higher than the average of all association constants of either ofthe initial pools.

Accessibility of the Target RNA; Computer-Supported Prediction of theSecondary Structure of the Target RNA

The complete secondary structure of the pregenomic RNA was predeterminedusing the program “MFold” (HUSAR: Heidelberg Unix Sequence AnalysisResources). The results of the in vitro selection experiments agree withthe accessibility of the target RNA. Thus DNA oligonucleotide analogswere obtained from short selected antisense RNA molecules and werestudied in transient in vivo replication tests.

121 1 20 DNA Artificial Sequence Description of Artificial Sequenceantisense nucleic acid against RNA originating from HBV 1 gcgagggagttcttcttcta 20 2 18 DNA Artificial Sequence Description of ArtificialSequence antisense nucleic acid against RNA originating from HBV 2gatcccccta gaaaattg 18 3 22 DNA Artificial Sequence Description ofArtificial Sequence antisense nucleic acid against RNA originating fromHBV 3 ggatcggcag aggagccaca aa 22 4 56 RNA Artificial SequenceDescription of Artificial Sequence antisense nucleic acid against RNAoriginating from HBV 4 gaauacucaa gcuaugcauc caacgcguug ggagcucuccggaucccccu agaaaa 56 5 57 RNA Artificial Sequence Description ofArtificial Sequence antisense nucleic acid against RNA originating fromHBV 5 gaauacucaa gcuaugcauc caacgcguug ggagcucucc ggaucccccu agaaaau 576 58 RNA Artificial Sequence Description of Artificial Sequenceantisense nucleic acid against RNA originating from HBV 6 gaauacucaagcuaugcauc caacgcguug ggagcucucc ggaucccccu agaaaauu 58 7 59 RNAArtificial Sequence Description of Artificial Sequence antisense nucleicacid against RNA originating from HBV 7 gaauacucaa gcuaugcauc caacgcguugggagcucucc ggaucccccu agaaaauug 59 8 60 RNA Artificial SequenceDescription of Artificial Sequence antisense nucleic acid against RNAoriginating from HBV 8 gaauacucaa gcuaugcauc caacgcguug ggagcucuccggaucccccu agaaaauuga 60 9 77 RNA Artificial Sequence Description ofArtificial Sequence antisense nucleic acid against RNA originating fromHBV 9 gaauacucaa gcuaugcauc caacgcguug ggagcucucc ggaucccccu agaaaauuga60 gagaagucca ccacgag 77 10 78 RNA Artificial Sequence Description ofArtificial Sequence antisense nucleic acid against RNA originating fromHBV 10 gaauacucaa gcuaugcauc caacgcguug ggagcucucc ggaucccccu agaaaauuga60 gagaagucca ccacgagu 78 11 79 RNA Artificial Sequence Description ofArtificial Sequence antisense nucleic acid against RNA originating fromHBV 11 gaauacucaa gcuaugcauc caacgcguug ggagcucucc ggaucccccu agaaaauuga60 gagaagucca ccacgaguc 79 12 80 RNA Artificial Sequence Description ofArtificial Sequence antisense nucleic acid against RNA originating fromHBV 12 gaauacucaa gcuaugcauc caacgcguug ggagcucucc ggaucccccu agaaaauuga60 gagaagucca ccacgagucu 80 13 81 RNA Artificial Sequence Description ofArtificial Sequence antisense nucleic acid against RNA originating fromHBV 13 gaauacucaa gcuaugcauc caacgcguug ggagcucucc ggaucccccu agaaaauuga60 gagaagucca ccacgagucu a 81 14 82 RNA Artificial Sequence Descriptionof Artificial Sequence antisense nucleic acid against RNA originatingfrom HBV 14 15 83 RNA Artificial Sequence Description of ArtificialSequence antisense nucleic acid against RNA originating from HBV 15gaauacucaa gcuaugcauc caacgcguug ggagcucucc ggaucccccu agaaaauuga 60gagaagucca ccacgagucu aga 83 16 84 RNA Artificial Sequence Descriptionof Artificial Sequence antisense nucleic acid against RNA originatingfrom HBV 16 gaauacucaa gcuaugcauc caacgcguug ggagcucucc ggaucccccuagaaaauuga 60 gagaagucca ccacgagucu agac 84 17 101 RNA ArtificialSequence Description of Artificial Sequence antisense nucleic acidagainst RNA originating from HBV 17 gaauacucaa gcuaugcauc caacgcguugggagcucucc ggaucccccu agaaaauuga 60 gagaagucca ccacgagucu agacucugcgguauugugag g 101 18 102 RNA Artificial Sequence Description ofArtificial Sequence antisense nucleic acid against RNA originating fromHBV 18 gaauacucaa gcuaugcauc caacgcguug ggagcucucc ggaucccccu agaaaauuga60 gagaagucca ccacgagucu agacucugcg guauugugag ga 102 19 103 RNAArtificial Sequence Description of Artificial Sequence antisense nucleicacid against RNA originating from HBV 19 gaauacucaa gcuaugcauccaacgcguug ggagcucucc ggaucccccu agaaaauuga 60 gagaagucca ccacgagucuagacucugcg guauugugag gau 103 20 104 RNA Artificial Sequence Descriptionof Artificial Sequence antisense nucleic acid against RNA originatingfrom HBV 20 gaauacucaa gcuaugcauc caacgcguug ggagcucucc ggaucccccuagaaaauuga 60 gagaagucca ccacgagucu agacucugcg guauugugag gauu 104 21113 RNA Artificial Sequence Description of Artificial Sequence antisensenucleic acid against RNA originating from HBV 21 gaauacucaa gcuaugcauccaacgcguug ggagcucucc ggaucccccu agaaaauuga 60 gagaagucca ccacgagucuagacucugcg guauugugag gauucuuguc aac 113 22 114 RNA Artificial SequenceDescription of Artificial Sequence antisense nucleic acid against RNAoriginating from HBV 22 gaauacucaa gcuaugcauc caacgcguug ggagcucuccggaucccccu agaaaauuga 60 gagaagucca ccacgagucu agacucugcg guauugugaggauucuuguc aaca 114 23 115 RNA Artificial Sequence Description ofArtificial Sequence antisense nucleic acid against RNA originating fromHBV 23 gaauacucaa gcuaugcauc caacgcguug ggagcucucc ggaucccccu agaaaauuga60 gagaagucca ccacgagucu agacucugcg guauugugag gauucuuguc aacaa 115 24116 RNA Artificial Sequence Description of Artificial Sequence antisensenucleic acid against RNA originating from HBV 24 gaauacucaa gcuaugcauccaacgcguug ggagcucucc ggaucccccu agaaaauuga 60 gagaagucca ccacgagucuagacucugcg guauugugag gauucuuguc aacaag 116 25 117 RNA ArtificialSequence Description of Artificial Sequence antisense nucleic acidagainst RNA originating from HBV 25 gaauacucaa gcuaugcauc caacgcguugggagcucucc ggaucccccu agaaaauuga 60 gagaagucca ccacgagucu agacucugcgguauugugag gauucuuguc aacaaga 117 26 137 RNA Artificial SequenceDescription of Artificial Sequence antisense nucleic acid against RNAoriginating from HBV 26 gaauacucaa gcuaugcauc caacgcguug ggagcucuccggaucccccu agaaaauuga 60 gagaagucca ccacgagucu agacucugcg guauugugaggauucuuguc aacaagaaaa 120 accccgccug uaacacg 137 27 138 RNA ArtificialSequence Description of Artificial Sequence antisense nucleic acidagainst RNA originating from HBV 27 gaauacucaa gcuaugcauc caacgcguugggagcucucc ggaucccccu agaaaauuga 60 gagaagucca ccacgagucu agacucugcgguauugugag gauucuuguc aacaagaaaa 120 accccgccug uaacacga 138 28 139 RNAArtificial Sequence Description of Artificial Sequence antisense nucleicacid against RNA originating from HBV 28 gaauacucaa gcuaugcauccaacgcguug ggagcucucc ggaucccccu agaaaauuga 60 gagaagucca ccacgagucuagacucugcg guauugugag gauucuuguc aacaagaaaa 120 accccgccug uaacacgag 13929 140 RNA Artificial Sequence Description of Artificial Sequenceantisense nucleic acid against RNA originating from HBV 29 gaauacucaagcuaugcauc caacgcguug ggagcucucc ggaucccccu agaaaauuga 60 gagaaguccaccacgagucu agacucugcg guauugugag gauucuuguc aacaagaaaa 120 accccgccuguaacacgagc 140 30 149 RNA Artificial Sequence Description of ArtificialSequence antisense nucleic acid against RNA originating from HBV 30gaauacucaa gcuaugcauc caacgcguug ggagcucucc ggaucccccu agaaaauuga 60gagaagucca ccacgagucu agacucugcg guauugugag gauucuuguc aacaagaaaa 120accccgccug uaacacgagc agggguccu 149 31 150 RNA Artificial SequenceDescription of Artificial Sequence antisense nucleic acid against RNAoriginating from HBV 31 gaauacucaa gcuaugcauc caacgcguug ggagcucuccggaucccccu agaaaauuga 60 gagaagucca ccacgagucu agacucugcg guauugugaggauucuuguc aacaagaaaa 120 accccgccug uaacacgagc agggguccua 150 32 151RNA Artificial Sequence Description of Artificial Sequence antisensenucleic acid against RNA originating from HBV 32 gaauacucaa gcuaugcauccaacgcguug ggagcucucc ggaucccccu agaaaauuga 60 gagaagucca ccacgagucuagacucugcg guauugugag gauucuuguc aacaagaaaa 120 accccgccug uaacacgagcagggguccua g 151 33 152 RNA Artificial Sequence Description ofArtificial Sequence antisense nucleic acid against RNA originating fromHBV 33 gaauacucaa gcuaugcauc caacgcguug ggagcucucc ggaucccccu agaaaauuga60 gagaagucca ccacgagucu agacucugcg guauugugag gauucuuguc aacaagaaaa 120accccgccug uaacacgagc agggguccua gg 152 34 153 RNA Artificial SequenceDescription of Artificial Sequence antisense nucleic acid against RNAoriginating from HBV 34 gaauacucaa gcuaugcauc caacgcguug ggagcucuccggaucccccu agaaaauuga 60 gagaagucca ccacgagucu agacucugcg guauugugaggauucuuguc aacaagaaaa 120 accccgccug uaacacgagc agggguccua gga 153 35163 RNA Artificial Sequence Description of Artificial Sequence antisensenucleic acid against RNA originating from HBV 35 gaauacucaa gcuaugcauccaacgcguug ggagcucucc ggaucccccu agaaaauuga 60 gagaagucca ccacgagucuagacucugcg guauugugag gauucuuguc aacaagaaaa 120 accccgccug uaacacgagcagggguccua ggaauccuga ugu 163 36 164 RNA Artificial Sequence Descriptionof Artificial Sequence antisense nucleic acid against RNA originatingfrom HBV 36 gaauacucaa gcuaugcauc caacgcguug ggagcucucc ggaucccccuagaaaauuga 60 gagaagucca ccacgagucu agacucugcg guauugugag gauucuugucaacaagaaaa 120 accccgccug uaacacgagc agggguccua ggaauccuga ugug 164 37165 RNA Artificial Sequence Description of Artificial Sequence antisensenucleic acid against RNA originating from HBV 37 gaauacucaa gcuaugcauccaacgcguug ggagcucucc ggaucccccu agaaaauuga 60 gagaagucca ccacgagucuagacucugcg guauugugag gauucuuguc aacaagaaaa 120 accccgccug uaacacgagcagggguccua ggaauccuga uguga 165 38 166 RNA Artificial SequenceDescription of Artificial Sequence antisense nucleic acid against RNAoriginating from HBV 38 gaauacucaa gcuaugcauc caacgcguug ggagcucuccggaucccccu agaaaauuga 60 gagaagucca ccacgagucu agacucugcg guauugugaggauucuuguc aacaagaaaa 120 accccgccug uaacacgagc agggguccua ggaauccugaugugau 166 39 167 RNA Artificial Sequence Description of ArtificialSequence antisense nucleic acid against RNA originating from HBV 39gaauacucaa gcuaugcauc caacgcguug ggagcucucc ggaucccccu agaaaauuga 60gagaagucca ccacgagucu agacucugcg guauugugag gauucuuguc aacaagaaaa 120accccgccug uaacacgagc agggguccua ggaauccuga ugugaug 167 40 168 RNAArtificial Sequence Description of Artificial Sequence antisense nucleicacid against RNA originating from HBV 40 gaauacucaa gcuaugcauccaacgcguug ggagcucucc ggaucccccu agaaaauuga 60 gagaagucca ccacgagucuagacucugcg guauugugag gauucuuguc aacaagaaaa 120 accccgccug uaacacgagcagggguccua ggaauccuga ugugaugu 168 41 217 RNA Artificial SequenceDescription of Artificial Sequence antisense nucleic acid against RNAoriginating from HBV 41 gaauacucaa gcuaugcauc caacgcguug ggagcucuccggaucccccu agaaaauuga 60 gagaagucca ccacgagucu agacucugcg guauugugaggauucuuguc aacaagaaaa 120 accccgccug uaacacgagc agggguccua ggaauccugaugugauguuc uccauguucg 180 ucacaggguc cccaguccuc gcggagauug acgagau 21742 218 RNA Artificial Sequence Description of Artificial Sequenceantisense nucleic acid against RNA originating from HBV 42 gaauacucaagcuaugcauc caacgcguug ggagcucucc ggaucccccu agaaaauuga 60 gagaaguccaccacgagucu agacucugcg guauugugag gauucuuguc aacaagaaaa 120 accccgccuguaacacgagc agggguccua ggaauccuga ugugauguuc uccauguucg 180 ucacaggguccccaguccuc gcggagauug acgagaug 218 43 219 RNA Artificial SequenceDescription of Artificial Sequence antisense nucleic acid against RNAoriginating from HBV 43 gaauacucaa gcuaugcauc caacgcguug ggagcucuccggaucccccu agaaaauuga 60 gagaagucca ccacgagucu agacucugcg guauugugaggauucuuguc aacaagaaaa 120 accccgccug uaacacgagc agggguccua ggaauccugaugugauguuc uccauguucg 180 ucacaggguc cccaguccuc gcggagauug acgagaugu 21944 220 RNA Artificial Sequence Description of Artificial Sequenceantisense nucleic acid against RNA originating from HBV 44 gaauacucaagcuaugcauc caacgcguug ggagcucucc ggaucccccu agaaaauuga 60 gagaaguccaccacgagucu agacucugcg guauugugag gauucuuguc aacaagaaaa 120 accccgccuguaacacgagc agggguccua ggaauccuga ugugauguuc uccauguucg 180 ucacaggguccccaguccuc gcggagauug acgagaugug 220 45 221 RNA Artificial SequenceDescription of Artificial Sequence antisense nucleic acid against RNAoriginating from HBV 45 gaauacucaa gcuaugcauc caacgcguug ggagcucuccggaucccccu agaaaauuga 60 gagaagucca ccacgagucu agacucugcg guauugugaggauucuuguc aacaagaaaa 120 accccgccug uaacacgagc agggguccua ggaauccugaugugauguuc uccauguucg 180 ucacaggguc cccaguccuc gcggagauug acgagaugug a221 46 222 RNA Artificial Sequence Description of Artificial Sequenceantisense nucleic acid against RNA originating from HBV 46 gaauacucaagcuaugcauc caacgcguug ggagcucucc ggaucccccu agaaaauuga 60 gagaaguccaccacgagucu agacucugcg guauugugag gauucuuguc aacaagaaaa 120 accccgccuguaacacgagc agggguccua ggaauccuga ugugauguuc uccauguucg 180 ucacaggguccccaguccuc gcggagauug acgagaugug ag 222 47 223 RNA Artificial SequenceDescription of Artificial Sequence antisense nucleic acid against RNAoriginating from HBV 47 gaauacucaa gcuaugcauc caacgcguug ggagcucuccggaucccccu agaaaauuga 60 gagaagucca ccacgagucu agacucugcg guauugugaggauucuuguc aacaagaaaa 120 accccgccug uaacacgagc agggguccua ggaauccugaugugauguuc uccauguucg 180 ucacaggguc cccaguccuc gcggagauug acgagaugugaga 223 48 224 RNA Artificial Sequence Description of ArtificialSequence antisense nucleic acid against RNA originating from HBV 48gaauacucaa gcuaugcauc caacgcguug ggagcucucc ggaucccccu agaaaauuga 60gagaagucca ccacgagucu agacucugcg guauugugag gauucuuguc aacaagaaaa 120accccgccug uaacacgagc agggguccua ggaauccuga ugugauguuc uccauguucg 180ucacaggguc cccaguccuc gcggagauug acgagaugug agag 224 49 238 RNAArtificial Sequence Description of Artificial Sequence antisense nucleicacid against RNA originating from HBV 49 gaauacucaa gcuaugcauccaacgcguug ggagcucucc ggaucccccu agaaaauuga 60 gagaagucca ccacgagucuagacucugcg guauugugag gauucuuguc aacaagaaaa 120 accccgccug uaacacgagcagggguccua ggaauccuga ugugauguuc uccauguucg 180 ucacaggguc cccaguccucgcggagauug acgagaugug agaggcaaua uucggagc 238 50 239 RNA ArtificialSequence Description of Artificial Sequence antisense nucleic acidagainst RNA originating from HBV 50 gaauacucaa gcuaugcauc caacgcguugggagcucucc ggaucccccu agaaaauuga 60 gagaagucca ccacgagucu agacucugcgguauugugag gauucuuguc aacaagaaaa 120 accccgccug uaacacgagc agggguccuaggaauccuga ugugauguuc uccauguucg 180 ucacaggguc cccaguccuc gcggagauugacgagaugug agaggcaaua uucggagca 239 51 240 RNA Artificial SequenceDescription of Artificial Sequence antisense nucleic acid against RNAoriginating from HBV 51 gaauacucaa gcuaugcauc caacgcguug ggagcucuccggaucccccu agaaaauuga 60 gagaagucca ccacgagucu agacucugcg guauugugaggauucuuguc aacaagaaaa 120 accccgccug uaacacgagc agggguccua ggaauccugaugugauguuc uccauguucg 180 ucacaggguc cccaguccuc gcggagauug acgagaugugagaggcaaua uucggagcag 240 52 241 RNA Artificial Sequence Description ofArtificial Sequence antisense nucleic acid against RNA originating fromHBV 52 gaauacucaa gcuaugcauc caacgcguug ggagcucucc ggaucccccu agaaaauuga60 gagaagucca ccacgagucu agacucugcg guauugugag gauucuuguc aacaagaaaa 120accccgccug uaacacgagc agggguccua ggaauccuga ugugauguuc uccauguucg 180ucacaggguc cccaguccuc gcggagauug acgagaugug agaggcaaua uucggagcag 240 g241 53 242 RNA Artificial Sequence Description of Artificial Sequenceantisense nucleic acid against RNA originating from HBV 53 gaauacucaagcuaugcauc caacgcguug ggagcucucc ggaucccccu agaaaauuga 60 gagaaguccaccacgagucu agacucugcg guauugugag gauucuuguc aacaagaaaa 120 accccgccuguaacacgagc agggguccua ggaauccuga ugugauguuc uccauguucg 180 ucacaggguccccaguccuc gcggagauug acgagaugug agaggcaaua uucggagcag 240 gg 242 54 243RNA Artificial Sequence Description of Artificial Sequence antisensenucleic acid against RNA originating from HBV 54 gaauacucaa gcuaugcauccaacgcguug ggagcucucc ggaucccccu agaaaauuga 60 gagaagucca ccacgagucuagacucugcg guauugugag gauucuuguc aacaagaaaa 120 accccgccug uaacacgagcagggguccua ggaauccuga ugugauguuc uccauguucg 180 ucacaggguc cccaguccucgcggagauug acgagaugug agaggcaaua uucggagcag 240 ggu 243 55 244 RNAArtificial Sequence Description of Artificial Sequence antisense nucleicacid against RNA originating from HBV 55 gaauacucaa gcuaugcauccaacgcguug ggagcucucc ggaucccccu agaaaauuga 60 gagaagucca ccacgagucuagacucugcg guauugugag gauucuuguc aacaagaaaa 120 accccgccug uaacacgagcagggguccua ggaauccuga ugugauguuc uccauguucg 180 ucacaggguc cccaguccucgcggagauug acgagaugug agaggcaaua uucggagcag 240 gguu 244 56 245 RNAArtificial Sequence Description of Artificial Sequence antisense nucleicacid against RNA originating from HBV 56 gaauacucaa gcuaugcauccaacgcguug ggagcucucc ggaucccccu agaaaauuga 60 gagaagucca ccacgagucuagacucugcg guauugugag gauucuuguc aacaagaaaa 120 accccgccug uaacacgagcagggguccua ggaauccuga ugugauguuc uccauguucg 180 ucacaggguc cccaguccucgcggagauug acgagaugug agaggcaaua uucggagcag 240 gguuu 245 57 246 RNAArtificial Sequence Description of Artificial Sequence antisense nucleicacid against RNA originating from HBV 57 gaauacucaa gcuaugcauccaacgcguug ggagcucucc ggaucccccu agaaaauuga 60 gagaagucca ccacgagucuagacucugcg guauugugag gauucuuguc aacaagaaaa 120 accccgccug uaacacgagcagggguccua ggaauccuga ugugauguuc uccauguucg 180 ucacaggguc cccaguccucgcggagauug acgagaugug agaggcaaua uucggagcag 240 gguuua 246 58 247 RNAArtificial Sequence Description of Artificial Sequence antisense nucleicacid against RNA originating from HBV 58 gaauacucaa gcuaugcauccaacgcguug ggagcucucc ggaucccccu agaaaauuga 60 gagaagucca ccacgagucuagacucugcg guauugugag gauucuuguc aacaagaaaa 120 accccgccug uaacacgagcagggguccua ggaauccuga ugugauguuc uccauguucg 180 ucacaggguc cccaguccucgcggagauug acgagaugug agaggcaaua uucggagcag 240 gguuuac 247 59 248 RNAArtificial Sequence Description of Artificial Sequence antisense nucleicacid against RNA originating from HBV 59 gaauacucaa gcuaugcauccaacgcguug ggagcucucc ggaucccccu agaaaauuga 60 gagaagucca ccacgagucuagacucugcg guauugugag gauucuuguc aacaagaaaa 120 accccgccug uaacacgagcagggguccua ggaauccuga ugugauguuc uccauguucg 180 ucacaggguc cccaguccucgcggagauug acgagaugug agaggcaaua uucggagcag 240 gguuuacu 248 60 59 RNAArtificial Sequence Description of Artificial Sequence antisense nucleicacid against RNA originating from HBV 60 gaauacucaa gcuaugcauccaacgcguug ggagcucucc ggaucggcag aggagccac 59 61 60 RNA ArtificialSequence Description of Artificial Sequence antisense nucleic acidagainst RNA originating from HBV 61 gaauacucaa gcuaugcauc caacgcguugggagcucucc ggaucggcag aggagccaca 60 62 61 RNA Artificial SequenceDescription of Artificial Sequence antisense nucleic acid against RNAoriginating from HBV 62 gaauacucaa gcuaugcauc caacgcguug ggagcucuccggaucggcag aggagccaca 60 a 61 63 62 RNA Artificial Sequence Descriptionof Artificial Sequence antisense nucleic acid against RNA originatingfrom HBV 63 gaauacucaa gcuaugcauc caacgcguug ggagcucucc ggaucggcagaggagccaca 60 aa 62 64 63 RNA Artificial Sequence Description ofArtificial Sequence antisense nucleic acid against RNA originating fromHBV 64 gaauacucaa gcuaugcauc caacgcguug ggagcucucc ggaucggcag aggagccaca60 aag 63 65 64 RNA Artificial Sequence Description of ArtificialSequence antisense nucleic acid against RNA originating from HBV 65gaauacucaa gcuaugcauc caacgcguug ggagcucucc ggaucggcag aggagccaca 60aagg 64 66 65 RNA Artificial Sequence Description of Artificial Sequenceantisense nucleic acid against RNA originating from HBV 66 gaauacucaagcuaugcauc caacgcguug ggagcucucc ggaucggcag aggagccaca 60 aaggu 65 67 66RNA Artificial Sequence Description of Artificial Sequence antisensenucleic acid against RNA originating from HBV 67 gaauacucaa gcuaugcauccaacgcguug ggagcucucc ggaucggcag aggagccaca 60 aagguu 66 68 67 RNAArtificial Sequence Description of Artificial Sequence antisense nucleicacid against RNA originating from HBV 68 gaauacucaa gcuaugcauccaacgcguug ggagcucucc ggaucggcag aggagccaca 60 aagguuc 67 69 68 RNAArtificial Sequence Description of Artificial Sequence antisense nucleicacid against RNA originating from HBV 69 gaauacucaa gcuaugcauccaacgcguug ggagcucucc ggaucggcag aggagccaca 60 aagguucc 68 70 72 RNAArtificial Sequence Description of Artificial Sequence antisense nucleicacid against RNA originating from HBV 70 gaauacucaa gcuaugcauccaacgcguug ggagcucucc ggaucggcag aggagccaca 60 aagguuccac gc 72 71 73RNA Artificial Sequence Description of Artificial Sequence antisensenucleic acid against RNA originating from HBV 71 gaauacucaa gcuaugcauccaacgcguug ggagcucucc ggaucggcag aggagccaca 60 aagguuccac gca 73 72 74RNA Artificial Sequence Description of Artificial Sequence antisensenucleic acid against RNA originating from HBV 72 gaauacucaa gcuaugcauccaacgcguug ggagcucucc ggaucggcag aggagccaca 60 aagguuccac gcau 74 73 75RNA Artificial Sequence Description of Artificial Sequence antisensenucleic acid against RNA originating from HBV 73 gaauacucaa gcuaugcauccaacgcguug ggagcucucc ggaucggcag aggagccaca 60 aagguuccac gcaug 75 74 76RNA Artificial Sequence Description of Artificial Sequence antisensenucleic acid against RNA originating from HBV 74 gaauacucaa gcuaugcauccaacgcguug ggagcucucc ggaucggcag aggagccaca 60 aagguuccac gcaugc 76 7577 RNA Artificial Sequence Description of Artificial Sequence antisensenucleic acid against RNA originating from HBV 75 gaauacucaa gcuaugcauccaacgcguug ggagcucucc ggaucggcag aggagccaca 60 aagguuccac gcaugcg 77 76102 RNA Artificial Sequence Description of Artificial Sequence antisensenucleic acid against RNA originating from HBV 76 gaauacucaa gcuaugcauccaacgcguug ggagcucucc ggaucggcag aggagccaca 60 aagguuccac gcaugcgcugauggccuaug gccaagcccc ag 102 77 103 RNA Artificial Sequence Descriptionof Artificial Sequence antisense nucleic acid against RNA originatingfrom HBV 77 gaauacucaa gcuaugcauc caacgcguug ggagcucucc ggaucggcagaggagccaca 60 aagguuccac gcaugcgcug auggccuaug gccaagcccc agc 103 78 104RNA Artificial Sequence Description of Artificial Sequence antisensenucleic acid against RNA originating from HBV 78 gaauacucaa gcuaugcauccaacgcguug ggagcucucc ggaucggcag aggagccaca 60 aagguuccac gcaugcgcugauggccuaug gccaagcccc agcc 104 79 105 RNA Artificial SequenceDescription of Artificial Sequence antisense nucleic acid against RNAoriginating from HBV 79 gaauacucaa gcuaugcauc caacgcguug ggagcucuccggaucggcag aggagccaca 60 aagguuccac gcaugcgcug auggccuaug gccaagccccagcca 105 80 106 RNA Artificial Sequence Description of ArtificialSequence antisense nucleic acid against RNA originating from HBV 80gaauacucaa gcuaugcauc caacgcguug ggagcucucc ggaucggcag aggagccaca 60aagguuccac gcaugcgcug auggccuaug gccaagcccc agccag 106 81 107 RNAArtificial Sequence Description of Artificial Sequence antisense nucleicacid against RNA originating from HBV 81 gaauacucaa gcuaugcauccaacgcguug ggagcucucc ggaucggcag aggagccaca 60 aagguuccac gcaugcgcugauggccuaug gccaagcccc agccagu 107 82 108 RNA Artificial SequenceDescription of Artificial Sequence antisense nucleic acid against RNAoriginating from HBV 82 gaauacucaa gcuaugcauc caacgcguug ggagcucuccggaucggcag aggagccaca 60 aagguuccac gcaugcgcug auggccuaug gccaagccccagccagug 108 83 109 RNA Artificial Sequence Description of ArtificialSequence antisense nucleic acid against RNA originating from HBV 83gaauacucaa gcuaugcauc caacgcguug ggagcucucc ggaucggcag aggagccaca 60aagguuccac gcaugcgcug auggccuaug gccaagcccc agccagugg 109 84 110 RNAArtificial Sequence Description of Artificial Sequence antisense nucleicacid against RNA originating from HBV 84 gaauacucaa gcuaugcauccaacgcguug ggagcucucc ggaucggcag aggagccaca 60 aagguuccac gcaugcgcugauggccuaug gccaagcccc agccaguggg 110 85 111 RNA Artificial SequenceDescription of Artificial Sequence antisense nucleic acid against RNAoriginating from HBV 85 gaauacucaa gcuaugcauc caacgcguug ggagcucuccggaucggcag aggagccaca 60 aagguuccac gcaugcgcug auggccuaug gccaagccccagccaguggg g 111 86 112 RNA Artificial Sequence Description ofArtificial Sequence antisense nucleic acid against RNA originating fromHBV 86 gaauacucaa gcuaugcauc caacgcguug ggagcucucc ggaucggcag aggagccaca60 aagguuccac gcaugcgcug auggccuaug gccaagcccc agccaguggg gg 112 87 113RNA Artificial Sequence Description of Artificial Sequence antisensenucleic acid against RNA originating from HBV 87 gaauacucaa gcuaugcauccaacgcguug ggagcucucc ggaucggcag aggagccaca 60 aagguuccac gcaugcgcugauggccuaug gccaagcccc agccaguggg ggu 113 88 114 RNA Artificial SequenceDescription of Artificial Sequence antisense nucleic acid against RNAoriginating from HBV 88 gaauacucaa gcuaugcauc caacgcguug ggagcucuccggaucggcag aggagccaca 60 aagguuccac gcaugcgcug auggccuaug gccaagccccagccaguggg gguu 114 89 115 RNA Artificial Sequence Description ofArtificial Sequence antisense nucleic acid against RNA originating fromHBV 89 gaauacucaa gcuaugcauc caacgcguug ggagcucucc ggaucggcag aggagccaca60 aagguuccac gcaugcgcug auggccuaug gccaagcccc agccaguggg gguug 115 90116 RNA Artificial Sequence Description of Artificial Sequence antisensenucleic acid against RNA originating from HBV 90 gaauacucaa gcuaugcauccaacgcguug ggagcucucc ggaucggcag aggagccaca 60 aagguuccac gcaugcgcugauggccuaug gccaagcccc agccaguggg gguugc 116 91 117 RNA ArtificialSequence Description of Artificial Sequence antisense nucleic acidagainst RNA originating from HBV 91 gaauacucaa gcuaugcauc caacgcguugggagcucucc ggaucggcag aggagccaca 60 aagguuccac gcaugcgcug auggccuauggccaagcccc agccaguggg gguugcg 117 92 118 RNA Artificial SequenceDescription of Artificial Sequence antisense nucleic acid against RNAoriginating from HBV 92 gaauacucaa gcuaugcauc caacgcguug ggagcucuccggaucggcag aggagccaca 60 aagguuccac gcaugcgcug auggccuaug gccaagccccagccaguggg gguugcgu 118 93 135 RNA Artificial Sequence Description ofArtificial Sequence antisense nucleic acid against RNA originating fromHBV 93 gaauacucaa gcuaugcauc caacgcguug ggagcucucc ggaucggcag aggagccaca60 aagguuccac gcaugcgcug auggccuaug gccaagcccc agccaguggg gguugcguca 120gcaaacacuu ggcac 135 94 136 RNA Artificial Sequence Description ofArtificial Sequence antisense nucleic acid against RNA originating fromHBV 94 gaauacucaa gcuaugcauc caacgcguug ggagcucucc ggaucggcag aggagccaca60 aagguuccac gcaugcgcug auggccuaug gccaagcccc agccaguggg gguugcguca 120gcaaacacuu ggcaca 136 95 137 RNA Artificial Sequence Description ofArtificial Sequence antisense nucleic acid against RNA originating fromHBV 95 gaauacucaa gcuaugcauc caacgcguug ggagcucucc ggaucggcag aggagccaca60 aagguuccac gcaugcgcug auggccuaug gccaagcccc agccaguggg gguugcguca 120gcaaacacuu ggcacag 137 96 138 RNA Artificial Sequence Description ofArtificial Sequence antisense nucleic acid against RNA originating fromHBV 96 gaauacucaa gcuaugcauc caacgcguug ggagcucucc ggaucggcag aggagccaca60 aagguuccac gcaugcgcug auggccuaug gccaagcccc agccaguggg gguugcguca 120gcaaacacuu ggcacaga 138 97 139 RNA Artificial Sequence Description ofArtificial Sequence antisense nucleic acid against RNA originating fromHBV 97 gaauacucaa gcuaugcauc caacgcguug ggagcucucc ggaucggcag aggagccaca60 aagguuccac gcaugcgcug auggccuaug gccaagcccc agccaguggg gguugcguca 120gcaaacacuu ggcacagac 139 98 140 RNA Artificial Sequence Description ofArtificial Sequence antisense nucleic acid against RNA originating fromHBV 98 gaauacucaa gcuaugcauc caacgcguug ggagcucucc ggaucggcag aggagccaca60 aagguuccac gcaugcgcug auggccuaug gccaagcccc agccaguggg gguugcguca 120gcaaacacuu ggcacagacc 140 99 141 RNA Artificial Sequence Description ofArtificial Sequence antisense nucleic acid against RNA originating fromHBV 99 gaauacucaa gcuaugcauc caacgcguug ggagcucucc ggaucggcag aggagccaca60 aagguuccac gcaugcgcug auggccuaug gccaagcccc agccaguggg gguugcguca 120gcaaacacuu ggcacagacc a 141 100 142 RNA Artificial Sequence Descriptionof Artificial Sequence antisense nucleic acid against RNA originatingfrom HBV 100 gaauacucaa gcuaugcauc caacgcguug ggagcucucc ggaucggcagaggagccaca 60 aagguuccac gcaugcgcug auggccuaug gccaagcccc agccaguggggguugcguca 120 gcaaacacuu ggcacagacc ag 142 101 151 RNA ArtificialSequence Description of Artificial Sequence antisense nucleic acidagainst RNA originating from HBV 101 gaauacucaa gcuaugcauc caacgcguugggagcucucc ggaucggcag aggagccaca 60 aagguuccac gcaugcgcug auggccuauggccaagcccc agccaguggg gguugcguca 120 gcaaacacuu ggcacagacc aggccguugc c151 102 152 RNA Artificial Sequence Description of Artificial Sequenceantisense nucleic acid against RNA originating from HBV 102 gaauacucaagcuaugcauc caacgcguug ggagcucucc ggaucggcag aggagccaca 60 aagguuccacgcaugcgcug auggccuaug gccaagcccc agccaguggg gguugcguca 120 gcaaacacuuggcacagacc aggccguugc cg 152 103 153 RNA Artificial Sequence Descriptionof Artificial Sequence antisense nucleic acid against RNA originatingfrom HBV 103 gaauacucaa gcuaugcauc caacgcguug ggagcucucc ggaucggcagaggagccaca 60 aagguuccac gcaugcgcug auggccuaug gccaagcccc agccaguggggguugcguca 120 gcaaacacuu ggcacagacc aggccguugc cga 153 104 154 RNAArtificial Sequence Description of Artificial Sequence antisense nucleicacid against RNA originating from HBV 104 gaauacucaa gcuaugcauccaacgcguug ggagcucucc ggaucggcag aggagccaca 60 aagguuccac gcaugcgcugauggccuaug gccaagcccc agccaguggg gguugcguca 120 gcaaacacuu ggcacagaccaggccguugc cgag 154 105 155 RNA Artificial Sequence Description ofArtificial Sequence antisense nucleic acid against RNA originating fromHBV 105 gaauacucaa gcuaugcauc caacgcguug ggagcucucc ggaucggcagaggagccaca 60 aagguuccac gcaugcgcug auggccuaug gccaagcccc agccaguggggguugcguca 120 gcaaacacuu ggcacagacc aggccguugc cgagc 155 106 156 RNAArtificial Sequence Description of Artificial Sequence antisense nucleicacid against RNA originating from HBV 106 gaauacucaa gcuaugcauccaacgcguug ggagcucucc ggaucggcag aggagccaca 60 aagguuccac gcaugcgcugauggccuaug gccaagcccc agccaguggg gguugcguca 120 gcaaacacuu ggcacagaccaggccguugc cgagca 156 107 196 RNA Artificial Sequence Description ofArtificial Sequence antisense nucleic acid against RNA originating fromHBV 107 gaauacucaa gcuaugcauc caacgcguug ggagcucucc ggaucggcagaggagccaca 60 aagguuccac gcaugcgcug auggccuaug gccaagcccc agccaguggggguugcguca 120 gcaaacacuu ggcacagacc aggccguugc cgagcaacgg gguaaagguucauguacugu 180 uuacuuagaa aggccu 196 108 197 RNA Artificial SequenceDescription of Artificial Sequence antisense nucleic acid against RNAoriginating from HBV 108 gaauacucaa gcuaugcauc caacgcguug ggagcucuccggaucggcag aggagccaca 60 aagguuccac gcaugcgcug auggccuaug gccaagccccagccaguggg gguugcguca 120 gcaaacacuu ggcacagacc aggccguugc cgagcaacgggguaaagguu cauguacugu 180 uuacuuagaa aggccuu 197 109 198 RNA ArtificialSequence Description of Artificial Sequence antisense nucleic acidagainst RNA originating from HBV 109 gaauacucaa gcuaugcauc caacgcguugggagcucucc ggaucggcag aggagccaca 60 aagguuccac gcaugcgcug auggccuauggccaagcccc agccaguggg gguugcguca 120 gcaaacacuu ggcacagacc aggccguugccgagcaacgg gguaaagguu cauguacugu 180 uuacuuagaa aggccuug 198 110 199 RNAArtificial Sequence Description of Artificial Sequence antisense nucleicacid against RNA originating from HBV 110 gaauacucaa gcuaugcauccaacgcguug ggagcucucc ggaucggcag aggagccaca 60 aagguuccac gcaugcgcugauggccuaug gccaagcccc agccaguggg gguugcguca 120 gcaaacacuu ggcacagaccaggccguugc cgagcaacgg gguaaagguu cauguacugu 180 uuacuuagaa aggccuugu 199111 200 RNA Artificial Sequence Description of Artificial Sequenceantisense nucleic acid against RNA originating from HBV 111 gaauacucaagcuaugcauc caacgcguug ggagcucucc ggaucggcag aggagccaca 60 aagguuccacgcaugcgcug auggccuaug gccaagcccc agccaguggg gguugcguca 120 gcaaacacuuggcacagacc aggccguugc cgagcaacgg gguaaagguu cauguacugu 180 uuacuuagaaaggccuugua 200 112 201 RNA Artificial Sequence Description of ArtificialSequence antisense nucleic acid against RNA originating from HBV 112gaauacucaa gcuaugcauc caacgcguug ggagcucucc ggaucggcag aggagccaca 60aagguuccac gcaugcgcug auggccuaug gccaagcccc agccaguggg gguugcguca 120gcaaacacuu ggcacagacc aggccguugc cgagcaacgg gguaaagguu cauguacugu 180uuacuuagaa aggccuugua a 201 113 202 RNA Artificial Sequence Descriptionof Artificial Sequence antisense nucleic acid against RNA originatingfrom HBV 113 gaauacucaa gcuaugcauc caacgcguug ggagcucucc ggaucggcagaggagccaca 60 aagguuccac gcaugcgcug auggccuaug gccaagcccc agccaguggggguugcguca 120 gcaaacacuu ggcacagacc aggccguugc cgagcaacgg gguaaagguucauguacugu 180 uuacuuagaa aggccuugua ag 202 114 203 RNA ArtificialSequence Description of Artificial Sequence antisense nucleic acidagainst RNA originating from HBV 114 gaauacucaa gcuaugcauc caacgcguugggagcucucc ggaucggcag aggagccaca 60 aagguuccac gcaugcgcug auggccuauggccaagcccc agccaguggg gguugcguca 120 gcaaacacuu ggcacagacc aggccguugccgagcaacgg gguaaagguu cauguacugu 180 uuacuuagaa aggccuugua agu 203 115204 RNA Artificial Sequence Description of Artificial Sequence antisensenucleic acid against RNA originating from HBV 115 gaauacucaa gcuaugcauccaacgcguug ggagcucucc ggaucggcag aggagccaca 60 aagguuccac gcaugcgcugauggccuaug gccaagcccc agccaguggg gguugcguca 120 gcaaacacuu ggcacagaccaggccguugc cgagcaacgg gguaaagguu cauguacugu 180 uuacuuagaa aggccuuguaaguu 204 116 242 RNA Artificial Sequence Description of ArtificialSequence antisense nucleic acid against RNA originating from HBV 116gaauacucaa gcuaugcauc caacgcguug ggagcucucc ggaucggcag aggagccaca 60aagguuccac gcaugcgcug auggccuaug gccaagcccc agccaguggg gguugcguca 120gcaaacacuu ggcacagacc aggccguugc cgagcaacgg gguaaagguu cauguacugu 180uuacuuagaa aggccuugua aguuggcgag aaagugaaag ccuguuuagc uuguauacau 240 gc242 117 256 RNA Artificial Sequence Description of Artificial Sequenceantisense nucleic acid against RNA originating from HBV 117 gaauacucaagcuaugcauc caacgcguug ggagcucucc ggaucggcag aggagccaca 60 aagguuccacgcaugcgcug auggccuaug gccaagcccc agccaguggg gguugcguca 120 gcaaacacuuggcacagacc aggccguugc cgagcaacgg gguaaagguu cauguacugu 180 uuacuuagaaaggccuugua aguuggcgag aaagugaaag ccuguuuagc uuguauacau 240 gcauacaaaggcauua 256 118 267 RNA Artificial Sequence Description of ArtificialSequence antisense nucleic acid against RNA originating from HBV 118gaauacucaa gcuaugcauc caacgcguug ggagcucucc ggaucggcag aggagccaca 60aagguuccac gcaugcgcug auggccuaug gccaagcccc agccaguggg gguugcguca 120gcaaacacuu ggcacagacc aggccguugc cgagcaacgg gguaaagguu cauguacugu 180uuacuuagaa aggccuugua aguuggcgag aaagugaaag ccuguuuagc uuguauacau 240gcauacaaag gcauuaaggc aggauau 267 119 293 RNA Artificial SequenceDescription of Artificial Sequence antisense nucleic acid against RNAoriginating from HBV 119 gaauacucaa gcuaugcauc caacgcguug ggagcucuccggaucggcag aggagccaca 60 aagguuccac gcaugcgcug auggccuaug gccaagccccagccaguggg gguugcguca 120 gcaaacacuu ggcacagacc aggccguugc cgagcaacgggguaaagguu cauguacugu 180 uuacuuagaa aggccuugua aguuggcgag aaagugaaagccuguuuagc uuguauacau 240 gcauacaaag gcauuaaggc aggauaucca cauuguguaaauggagcagc aaa 293 120 305 RNA Artificial Sequence Description ofArtificial Sequence antisense nucleic acid against RNA originating fromHBV 120 gaauacucaa gcuaugcauc caacgcguug ggagcucucc ggaucggcagaggagccaca 60 aagguuccac gcaugcgcug auggccuaug gccaagcccc agccaguggggguugcguca 120 gcaaacacuu ggcacagacc aggccguugc cgagcaacgg gguaaagguucauguacugu 180 uuacuuagaa aggccuugua aguuggcgag aaagugaaag ccuguuuagcuuguauacau 240 gcauacaaag gcauuaaggc aggauaucca cauuguguaa auggagcagcaaagcccaaa 300 agacc 305 121 352 RNA Artificial Sequence Description ofArtificial Sequence antisense nucleic acid against RNA originating fromHBV 121 gaauacucaa gcuaugcauc caacgcguug ggagcucucc ggaucggcagaggagccaca 60 aagguuccac gcaugcgcug auggccuaug gccaagcccc agccaguggggguugcguca 120 gcaaacacuu ggcacagacc aggccguugc cgagcaacgg gguaaagguucauguacugu 180 uuacuuagaa aggccuugua aguuggcgag aaagugaaag ccuguuuagcuuguauacau 240 gcauacaaag gcauuaaggc aggauaucca cauuguguaa auggagcagcaaagcccaaa 300 agacccacaa uucuuugaca uacuuuccaa ucaauaggcc uguuaacagg aa352

What is claimed is:
 1. An antisense nucleic acid complementary to an RNAmolecule derived from a hepatitis B virus, wherein the nucleic acid hasan association constant of at least 5×10³ M⁻¹ s⁻¹ and is capable ofreducing the expression of a hepatitis B virus polvpeptide in aninfected cell, and wherein the antisense nucleic acid is selected fromthe group consisting of SEQ ID NOS: 1-3.
 2. A vector assentiallycomprising a nucleic acid sequence complenentary to the antisensenucleic asid of claim 1.